Project Summary Montana Molecular is in the business of developing and marketing genetically encoded biosensors for academic research and drug discovery in living cells. Our biosensors provide robust detection of crucial second messenger signaling, including DAG, PIP2, cAMP and Ca2+ on both fluorescence microscopes and the large installed base of automated plate readers, with metrics that match or surpass those of traditional biochemical and luminescence based assays. We have an established customer base, with over 100 early adopters in academia and industry. Our customers and industry partners have indicated a growing need for assays capable of targeting specific populations of living neurons and for ?assay-ready? iPSC derived neuronal lines for use in academia and secondary drug screens. Genetically encoded assays for GPCR signaling, developed by Montana Molecular, fit seamlessly with these needs. They can be used for kinetic measurements in living cells, can be targeted to specific populations of cells in complex tissue, and have been independently validated on a wide variety of fluorescence microscopes and automated plate readers. To meet the needs of our customers and to penetrate the growing human iPSC market, we propose two specific aims. In Aim 1 we will engineer a new baculovirus vector that circumvents the payload limits and safety considerations of Lentivirus and AAV. In Aim 2, we will partner with iPSC developers in both academia and industry to create the first assay-ready iPSC derived neurons stably expressing biosensors for the detection of multiple facets of GPCR signaling.